Novel polypeptides, DNAs encoding the same and use thereof

ABSTRACT

Polypeptide produced from human stromal cell line, the process for the preparation of the polypeptide, DNA encoding the polypeptide, vector carrying the DNA, host cell transformed by the vector, antibody of the polypeptide, and pharmaceutical composition containing the polypeptide or the antibody.

TECHNICAL FIELD

[0001] The invention is related to novel polypeptides produced by acertain human stromal cell line and DNAs encoding the said polypeptides.

[0002] More particularly, the invention is related to novel polypeptidesnamed to OAF065α and OAF065β (called them OAF065s hereafter), a processfor the preparation them, DNAs encoding the said polypeptides, a vectorcontaining the polypeptide, a host cell transformed by the vector,antibody of the said polypeptide, a pharmaceutical compositioncontaining the polypeptide or antibody.

TECHNICAL BACKGROUND

[0003] It is known that bone marrow stromal cells form bone marrow microenvironment of immunologic, hematopoietic system etc, and they produceand secret essential factors to induce of proliferation anddifferentiation of stem cells, e.g. IL-7, SCF, IL-11, M-CSF, G-CSF,GM-CSF, IL-6, TGF-β, LIF etc. It is also made clear that a certain bonemarrow stromal cells are related to bone metabolism (Kenneth DorshkindAnnu. Rev. Immunol. 8, 111-137.1990). However, roles of stromal cell arenot reconstituted completely from only isolated factors yet. It maysuggest that existence of any factors which are not isolated yet.

DISCLOSURE OF THE INVENTION

[0004] The present inventors have directed their attention to this pointand energetic research has been carried out in order to find novelfactors (polypeptides) especially secretory and membrane protein whichare generated by a certain stromal cells.

[0005] Until now, when a man skilled in the art intends to obtain aparticular polypeptide or a DNA encoding it, he generally utilizesmethods by confirming an intended biological activity in a tissue or ina cell medium, isolating and purifying the polypeptide and then cloninga gene or methods by “expression-cloning” with the guidance of thebiological activity.

[0006] However, physiologically active polypeptides in living body haveoften many kinds of activities. Therefore, it is increasing that after agene is cloned, the gene is found to be identical to that encoding apolypeptide already known. Generally bone marrow stromal cell generatesonly a very slight amount of a factor and it makes difficult to isolateand to purify the factor and to confirm its biological activity.

[0007] Recent rapid developments in techniques for constructing cDNAsand sequencing techniques have made it possible to quickly sequence alarge amount of cDNAs. By utilizing these techniques, a process, whichcomprises constructing cDNAs at random, identifying the nucleotidesequences thereof, expressing novel polypeptides encoded by them, is nowin progress. Although this process is advantageous in that a gene can becloned and information regarding its nucleotide sequence can be obtainedwithout any biochemical or genetic analysis, the target gene can bediscovered thereby only accidentally in many cases.

[0008] The present inventors have studied cloning method of genes codingproliferation and/or differentiation factors functioning inhematopoietic systems and immune systems. Focusing their attention onthe fact that most of the secretory proteins such as proliferationand/or differentiation factors (for example various cytokines) andmembrane proteins such as receptors thereof (hereafter these proteinswill be referred to generally as secretory proteins and the like) havesequences called signal peptides in the N-termini, the inventorsconducted extensive studies on a process for efficiently and selectivelycloning a gene coding for a signal peptide. Finally, we havesuccessfully invented a screening method for cDNAs having sequenceencoding signal peptides, we called the method as signal sequence trap(SST) (See Japanese Patent Application No. 6-13951). We also developedyeast SST method on the same concept. By the method using yeast, genesincluding sequence encoding signal peptide can be identified more easilyand effectively (See U.S. Pat. No. 5,536,637).

[0009] By using SST method, the present inventors achieved to find novelmembrane proteins produced by bone marrow stromal cell and DNAs encodingthem, and we then completed the invention. The polypeptide OAF065s ofthe invention are not known one, when amino acid sequences of thepolypeptide was compared by a computer to all known sequences in database of Swiss Prot Release 33. It was found out that the polypeptides ofthe invention are type-I membrane protein and they have extracellularCys rich region which commonly exists in the receptor family of Tumornecrosis factor (TNF) (See FIG. 1). So it was suggested that thepolypeptides of the invention are novel membrane proteins which belongto TNF receptor family.

BRIEF DESCRIPTION OF THE DRAWING

[0010]FIG. 1 shows comparison of the amino acid sequence of theinvention and that of TNF receptor family. hTNFR1 represents humannecrosis factor receptor 1, hTNFR2 represents human necrosis factorreceptor 2, hNGFR represents human nerve growth factor receptor, andhFas represents human Fas, in this figure.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The invention provides:

[0012] 1) a polypeptide comprising an amino acid sequence shown in SEQID NO. 1 or NO. 5,

[0013] 2) a DNA encoding the polypeptides described above (1),

[0014] 3) a DNA comprising a nucleotide sequence shown in SEQ ID NO. 2or NO. 6,

[0015] 4) a DNA comprising a nucleotide sequence shown in SEQ ID NO. 3or NO. 7.

[0016] More particularly, the invention is concerned with a polypeptidecomprising amino acid sequence shown in SEQ ID NO. 1 or 5 insubstantially purified form, a homologue thereof, a fragment of thesequence and a homologue of the fragment. Further, the invention isconcerned with DNAs encoding the above peptides. More particularly theinvention is provided DNAs comprising nucleotide sequence shown in SEQID NO. 2, 3, 6 or 7, and DNA containing a fragment which is selectivelyhybridizing to the DNA comprising nucleotide sequence shown in SEQ IDNO. 2, 3, 6 or 7.

[0017] A polypeptide comprising amino acid sequence shown in SEQ ID NO.1 or 5 in substantially purified form will generally comprise thepolypeptide in a preparation in which more than 90%, e.g. 95%, 98% or99% of the polypeptide in the preparation is that of the SEQ ID NO. 1 or5. A homologue of polypeptide comprising amino acid sequence shown inSEQ ID NO. 1 or 5 will be generally at least 70%, preferably at least 80or 90% and more preferably at least 95% homologous to the polypeptidecomprising amino acid sequence shown in SEQ ID NO. 1 over a region of atleast 20, preferably at least 30, for instance 40, 60 or 100 morecontiguous amino acids. Such a polypeptide homologue will be referred toa polypeptide of the invention.

[0018] Generally, a fragment of polypeptide comprising amino acidsequence shown in SEQ ID NO. 1 or 5 or its homologues will be at least10, preferably at least 15, for example 20, 25, 30, 40, 50 or 60 aminoacids in length, and are also referred to by the term “a polypeptide ofthe invention”.

[0019] A DNA capable of selectively hybridizing to the DNA comprisingnucleotide sequence shown in SEQ ID NO. 2, 3, 6 or 7 will be generallyat least 70%, preferably at least 80 or 90% and more preferably at least95%. homologous to the DNA comprising nucleotide sequence shown in SEQID NO. 2, 3, 6 or 7 over a region of at least 20, preferably at least30, for instance 40, 60 or 100 or more contiguous nucleotides. Such DNAwill be referred to “a cDNA of the invention”.

[0020] Fragments of the DNA comprising nucleotide sequence shown in SEQID NO. 2, 3, 6 or 7 will be at least 10, preferably at least 15, forexample 20, 25, 30 or 40 nucleotides in length, and will be alsoreferred to “a DNA of the invention” as used herein.

[0021] A further embodiment of the invention provides replication andexpression vectors carrying DNA of the invention. The vectors may be,for example, plasmid, virus or phage vectors provided with an origin ofreplication, optionally a promoter for the expression of the said DNAand optionally a regulator of the promoter. The vector may contain oneor more selectable marker genes, for example a ampicillin resistancegene. The vector may be used in vitro, for example of the production ofRNA corresponding to the cDNA, or used to transfect or transfect a hostcell.

[0022] A further embodiment of the invention provides host cellstransformed with the vectors for the replication and expression of theDNA of the invention, including the DNA SEQ ID NO. 2, 3, 6 or 7 or theopen reading frame thereof. The cells will be chosen to be compatiblewith the vector and may for example be bacterial, yeast, insect ormammalian.

[0023] A further embodiment of the invention provides a method ofproducing a polypeptide which comprises culturing host cells of theinvention under conditions effective to express a polypeptide of theinvention. Preferably, in addition, such a method is carried out underconditions in which the polypeptide of the invention is expressed andthen produced from the host cells.

[0024] DNA of the invention may also be inserted into the vectorsdescribed above in an antisense orientation in order to proved for theproduction of antisense RNA. Such antisense RNA may be used in a methodof controlling the levels of a polypeptide of the invention in a cell.

[0025] The invention also provides monoclonal or polyclonal antibodiesagainst a polypeptide of the invention. The invention further provides aprocess for the production of monoclonal or polyclonal antibodies to thepolypeptides of the invention. Monoclonal antibodies may be prepared bycommon hybridoma technology using polypeptides of the invention orfragments thereof, as an immunogen. Polyclonal antibodies may also beprepared by common means which comprise inoculating host animals, forexample a rat or a rabbit, with polypeptides of the invention andrecovering immune serum.

[0026] The invention also provides pharmaceutical compositionscontaining a polypeptide of the invention, or an antibody thereof, inassociation with a pharmaceutically acceptable diluent and/or carrier.

[0027] The polypeptide of the invention includes that which a part oftheir amino acid sequence is lacking (e.g., a polypeptide comprised ofthe only essential sequence for revealing a biological activity in anamino acid sequence shown in SEQ ID NO.1), that which a part of theiramino acid sequence is replaced by other amino acids (e.g., thosereplaced by an amino acid having a similar property) and that whichother amino acids are added or inserted into a part of their amino acidsequence, as well as those comprising the amino acid sequence shown inSEQ ID NO. 1 or 5.

[0028] As known well, there are one to six kinds of codon as thatencoding one amino acid (for example, one kind of codon for Methioine(Met), and six kinds of codon for leucine (Leu) are known). Accordingly,the nucleotide sequence of DNA can be changed in order to encode thepolypeptide having the same amino acid sequence.

[0029] The DNA of the invention, specified in (2) includes a group ofevery nucleotide sequences encoding polypeptides (1) shown in SEQ ID NO.1 or 5. There is a probability that yield of a polypeptide is improvedby changing a nucleotide sequence.

[0030] The DNA specified in (3) is the embodiment of the DNA shown in(2), and indicate the sequence of natural form.

[0031] The DNA shown in (4) indicates the sequence of the DNA specifiedin (3) with natural non-translational region. cDNA carrying nucleotidesequence shown in SEQ ID NO. 3 is prepared by the following method:

[0032] Brief description of Yeast SST method (see U.S. Pat. No.5,536,637) is as follows.

[0033] Yeast such as Saccharomyces cerevisiae should secrete invertaseinto the medium in order to take sucrose or raffinose as a source ofenergy or carbon (Invertase is an enzyme to cleave raffinose intosucrose and melibiose, sucrose into fructose and glucose.). It is knownthat many known mammalian signal sequence make yeast secrete itsinvertase. From these knowledge, SST method was developed as a screeningmethod to find novel signal sequence which make it possible can tosecrete yeast invertase from mammalian cDNA library. SST method usesyeast growth on raffinose medium as a marker. Non-secretory typeinvertase gene SUC2 (GENBANK Accession No. V 01311) lacking initiationcodon ATG was inserted to yeast expression vector to prepare yeast SSTvector pSUC2. In this expression vector, ADH promoter, ADH terminator(both were derived from AAH5 plasmid (Gammerer, Methods in Enzymol. 101,192-201, 1983)), 2μ ori (as a yeast replication origin), TRP1 (as ayeast selective marker), ColE1 ori(as a E. Coli replication origin) andampicillin resistance gene (as a drug resistance marker) were inserted.Mammalian cDNA was inserted into the upstream of SUC2 gene to prepareyeast SST cDNA library. Yeast lacking secretory type invertase, wastransformed with this library. If inserted mammalian cDNA encodes asignal peptide, yeast could be survive in raffinose medium as a resultof restoring secretion of invertase. Only to culture yeast colonies,prepare plasmids and determine the nucleotide sequence of the insertcDNAs, it is possible to identify novel signal peptide rapidly andeasily.

[0034] Preparation of yeast SST CDNA library is as follows:

[0035] (1) mRNA is isolated from the targeted cells, second-strandsynthesis is performed by using random primer with certain restrictionenzyme (enzyme I) recognition site,

[0036] (2) double-strand cDNA is ligated to adapter containing certainrestriction endonuclease (enzyme II) recognition site, differ fromenzyme I, digested with enzyme I and fractionated in a appropriate size,

[0037] (3) obtained cDNA fragment is inserted into yeast expressionvector on the upstream region of invertase gene which signal peptide isdeleted and the library was transformed.

[0038] Detailed description of each step is as follows:

[0039] (1) mRNA is isolated from mammalian organs and cell linesstimulate them with appropriate stimulator if necessary) by knownmethods (Molecular Cloning (Sambrook, J., Fritsch, E. F. and Maniatis,T., Cold Spring Harbor Laboratory Press, 1989) or Current Protocol inMolecular Biology (F. M. Ausubel et al, John Wiley & Sons, Inc.) if notremark especially).

[0040] HAS303 (human bone marrow stromal cell line: provide fromProfessor Keisuke Sotoyama, Dr. Makoto Aizawa of Tokyo Medical College,1st medicine; see J. Cell. Physiol., 148, 245-251, 1991 and ExperimentalHematol., 22, 482-487, 1994) and HUVEC (human umbilical vein cordendothelial cell: ATCC No. CRL-1730) are chosen as a tissue source.Double-strand cDNA synthesis using random primer is performed by knownmethods.

[0041] Any sites may be used as restriction endonuclease recognitionsite I which is linked to adapter and restriction endonucleaserecognition site II which is used in step (2), if both sites aredifferent each other. Preferably, EcoRI is used as enzyme I and XhoI asenzyme II.

[0042] In step (2), cDNA is created blunt-ends with T4 DNA polymerase,ligated enzyme II adapter and digested with enzyme I. Fragment CDNA isanalyzed with agarose-gel electrophoresis (AGE) and is selected CDNAfraction ranging in size from 300 to 800 bp. As mentioned above, anyenzyme may be used as enzyme II if it is not same the enzyme I.

[0043] In step (3), cDNA fragment obtained in step (2) is inserted intoyeast expression vector on the upstream region of invertase gene whichsignal peptide is deleted. E. coli transformed with the expressionvector. Many vectors are known as yeast expression plasmid vector. Forexample, YEp24 is also functioned in E. Coli. Preferably pSUC2 asdescribed above is used.

[0044] Many host E. Coli strains are known for transformation,preferably DH10B competent cell is used. Any known transformation methodis available, preferably it is performed by electropolation method.Transformant is cultured by conventional methods to obtain cDNA libraryfor yeast SST method.

[0045] However not every All of the clones do not contain cDNA fragment.Further all of the gene fragments do not encode unknown signal peptides.It is therefore necessary to screen a gene fragment encoding for anunknown signal peptide from the library.

[0046] Therefore, screening of fragments containing a sequence encodingan appropriate signal peptide is performed by transformation of the cDNAlibrary into Saccharomyces cerevisiae (e.g. YT455 strain) which lackinvertase (it may be prepared by known methods.). Transformation ofyeast is performed by known methods, e.g. lithium acetate method.Transformant is cultured in a selective medium, then transferred to amedium containing raffinose as a carbon source. Survival colonies areselected and then prepared plasmid. Survival colonies on araffinose-medium indicates that some signal peptide of secretory proteinwas inserted to this clone.

[0047] Isolated positive clones is determined the nucleotide sequence.As to a cDNA encodes unknown protein, full-length clone may be isolatedby using cDNA fragment as a probe and then determined to obtainfull-length nucleotide sequence. These manipulation is performed byknown methods.

[0048] Once the nucleotide sequences shown in SEQ ID NO. 2, 3, 6 or 7are determined partially or preferably fully, it is possible to obtainDNA encode mammalian protein itself, homologue or subset. cDNA libraryor mRNA derived from mammals was screened by PCR with any synthesizedoligonucleotide primers or by hybridization with any fragment as aprobe. It is possible to obtain DNA encodes other mammalian homologueprotein from other mammalian cDNA or genome library.

[0049] If a cDNA obtained above contains a nucleotide sequence of cDNAfragment obtained by SST (or consensus sequence thereof), it will bethought that the cDNA encodes signal peptide. So it is clear that thecDNA will be full-length or almost full. (All signal sequences exist atN-termini of a protein and are encoded at 5′-temini of open readingframe of cDNA.)

[0050] The confirmation may be carried out by Northern analysis with thesaid cDNA as a probe. It is thought that the cDNA is almost completelength, if length of the cDNA is almost the same length of the mRNAobtained in the hybridizing band.

[0051] Once the nucleotide sequences shown in SEQ ID NOs. 2, 3, 6 or 7are determined, DNAs of the invention are obtained by chemicalsynthesis, or by hybridization making use of nucleotide fragments whichare chemically synthesized as a probe. Furthermore, DNAs of theinvention are obtained in desired amount by transforming a vector thatcontains the DNA into a proper host, and culturing the transformant.

[0052] The polypeptides of the invention may be prepared by:

[0053] (1) isolating and purifying from an organism or a cultured cell,

[0054] (2) chemically synthesizing, or

[0055] (3) using recombinant DNA technology, preferably, by the methoddescribed in (3) in an industrial production.

[0056] Examples of expression system (host-vector system) for producinga polypeptide by using recombinant DNA technology are the expressionsystems of bacteria, yeast, insect cells and mammalian cells.

[0057] In the expression of the polypeptide, for example, in E. Coli,the expression vector is prepared by adding the initiation codon (ATG)to 5′ end of a DNA encoding mature peptide, connecting the DNA thusobtained to the downstream of a proper promoter (e.g., trp promoter, lacpromoter, λ PL promoter, T7 promoter etc.), and then inserting it into avector (e.g., pBR322, pUC18, pUC19 etc.) which functions in an E. colistrain.

[0058] Then, an E. coli strain (e.g., E. coli DH1 strain, E. coli JM109strain, E. coli HB101 strain, etc.) which is transformed with theexpression vector described above may be cultured in a appropriatemedium to obtain the desired polypeptide. When a signal peptide ofbacteria (e.g., signal peptide of pel B) is utilized, the desiredpolypeptide may be also released in periplasm. Furthermore, a fusionprotein with other polypeptide may be also produced easily.

[0059] In the expression of the polypeptide,.for example, in a mammaliancells, for example, the expression vector is prepared by inserting theDNA encoding nucleotide shown in SEQ ID NO. 3 or 7 into the downstreamof a proper promoter (e.g., SV40 promoter, LTR promoter, metallothioneinpromoter etc.) in a proper vector (e.g., retrovirus vector, papillomavirus vector, vaccinia virus vector, SV40 vector, etc.). A propermammalian cell (e.g., monkey COS-7 cell, Chinese hamster CHO cell, mouseL cell etc.) is transformed with the expression vector thus obtained,and then the transformant is cultured in a proper medium to get adesired polypeptide on the cell membrane. A vector described above canbe inserted with deletion mutant DNA that encodes sequence, which isdeleted transmembrane region from SEQ ID NOs. 3 or 7 and the expressionvector can be transfected into an appropriate mammalian cell. The aimedsoluble protein can be secreted into the culture medium. The polypeptideavailable by the way described above can be isolated and purified byconventional biochemical method.

INDUSTRIAL APPLICABILITY

[0060] The polypeptide OAF065s of the invention show significanthomology with a series of proteins which belong to TNF receptor family.Proteins, which belong to TNF receptor family, are type-1 membraneprotein which have 3 to 6 repeated structure containing 6 Cys residuesin the extracellular domain. It has been apparent that the proteins arerelated to proliferation, differentiation cell death of various cells bythe interaction with ligand thereof (Craig A. Smith et. al., Cell, 76,959-962, 1994). For instance, Neuronal growth factor (NGF) receptor/NGFare essential for keeping several kinds of neuronal cells surviving,allowing neuronal tubes to elongate and promoting to make neuronaltransmitters (Chao M. V., J. Neurobiol., 25, 1373-1385, 1994) Fas/FasLis essential for maintaining homeostasis in vivo, such as destruction ofcancer cells and removal of auto-reactive lymphocytes via itsapoptosis-inducing activity, and also relates to CD4-positive T cellreduction in AIDS, fulminant hepatitis, graft versus host disease (GVHD)after transplantation and the onset of various autoimmune diseases(Nagata S. et. al., Science, 267, 1449-1456, 1995). CD40/CD40L isessential for activating B cells (acceleration of growth and antibodyproduction) via T/B cell interaction (Banchereau J. et. al., Annu. Rev.Immunol., 12,881-922, 1994). TNF receptor/TNF and lymphotoxin (LT)receptor/LT have activities, such as growth, activation anddifferentiation induction of various immune and hematopoietic cells,cytotoxicity and growth inhibition of tumor cells, growth and activationof various connective tissues (e.g., endothelial cells, fibroblasts,osteoblasts, etc.) and viral growth inhibition, and are also essentialfor the morphology or organ formation of lymphoid tissue (Ware C. F. etal., Curr. Topics Microbiol. Immunol., 198, 175-218, 1995).

[0061] Since repetitive structures of Cys are present at three points inthe extracellular domain of the polypeptide of the invention, it isobvious that this is a novel protein belonging to the TNF receptorfamily and exerts its activity via a ligand belonging to a known orunknown TNF family. In consequence, it is considered that thepolypeptide of the invention and a CDNA molecule which encodes thepolypeptide will show one or more of the effects or biologicalactivities (including those which relates to the assays cited below)concerning differentiation, proliferation, growth, survival or celldeath of hematopoietic, immune and nerve system cells, immune systemfunctions, proliferation and growth of tumor, inflammations, bonemetabolism, etc. The effects or biological activities described inrelation to the polypeptide of the invention are provided byadministration or use of the polypeptide or by administration or use ofa cDNA molecule which encodes the polypeptide (e.g., vector suitable forgene therapy or cDNA introduction).

[0062] 1) Cytokine Activity and Cell Proliferation/DifferentiationActivity

[0063] The polypeptide of the invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother cytokines in certain cell populations. Many protein factorsdiscovered to date, including all known cytokines, have exhibitedactivity in one or more factor dependent cell proliferation assays, andhence the assays serve as a convenient confirmation of cytokineactivity. The activity of a polypeptide of the invention is evidenced byany one of a number of routine factor dependent cell proliferationassays for cell lines.

[0064] 2) Immune Stimulating/Suppressing Activity

[0065] The polypeptide of the invention may also exhibit immunestimulating or immune suppressing activity. The polypeptide of theinvention may be useful in the treatment of various immune deficienciesand disorders (including severe combined immunodeficiency (SCID)), e.g.,in regulating (up or down) growth and proliferation of T and/or Blymphocytes, as well as effecting the cytolytic activity of NK cells andother cell populations. These immune deficiencies may be genetic or becaused by viral (e.g. HIV) as well as bacterial or fungal infections, ormay result from autoimmune disorders. More specifically, infectiousdiseases causes by viral, bacterial, fungal or other infection may betreatable using the polypeptide of the invention, including infectionsby HIV, hepatitis viruses, herpes viruses, mycobacteria, leshmania,malaria and various fungal infections such as candida. Of course, inthis regard, a polypeptide of the invention may also be useful where aboost to the immune system generally would be indicated, i.e., in thetreatment of cancer.

[0066] Such a polypeptide of the invention may also to be useful in thetreatment of allergic reactions and conditions, such as asthma or otherrespiratory problems.

[0067] The polypeptide of the invention may also suppress chronic oracute inflammation, such as, for example, that associated with infection(such as septic shock or systemic inflammatory response syndrome(SIRS)), inflammatory bowel disease, Crohn's disease or resulting fromover production of cytokines such as TNF or IL-I (such as the effectdemonstrated by IL- 11).

[0068] 3) Hematopoiesis Regulating Activity

[0069] The polypeptide of the invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis.

[0070] The said biological activities are concerned with the followingall or some example(s). e.g. in supporting the growth and proliferationof erythroid progenitor cells alone or in combination with othercytokines, thereby indicating utility. for example, in treating variousanemias or for use in conjunction with irradiation/chemotherapy tostimulate the production of erythroid precursors and/or erythroid cells;

[0071] in supporting the growth and proliferation of myeloid cells suchas granulocytes and monocytes/macrophages (i.e., traditional CSFactivity) useful, for example, in conjunction with chemotherapy toprevent or treat consequent myelo-suppression;

[0072] in supporting the growth and proliferation of megakaryocytes andconsequently of platelets thereby allowing prevention or treatment ofvarious platelet disorders such as thrombocytopenia, and generally foruse in place of or complimentary to platelet transfusions;

[0073] and/or in supporting the growth and proliferation ofhematopoietic stem cells which are capable of maturing to any and all ofthe above-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e. in conjunction with bone marrow transplantation) as normalcells or genetically manipulated for gene therapy.

[0074] The activity of the polypeptide of the invention may, among othermeans. be measured by the following methods

[0075] 4) Tissue Generation/Regeneration Activity

[0076] The polypeptide of the invention also may have utility incompositions used for bone, cartilage, tendon, Ligament and/or nervetissue growth or regeneration, as well as for wound healing and tissuerepair, and in the treatment of bums, incisions and ulcers. Thepolypeptide of the invention, which induces cartilage and/or bone growthin circumstances where bone is not normally formed, has application inthe healing of bone fractures and cartilage damage or defects in humansand other animals. Such a preparation employing the polypeptide of theinvention may have prophylactic use in closed as well as open fracturereduction and also in the improved fixation of artificial joints. Denovo bone formation induced by an osteogenic agent contributes to therepair of congenital, trauma induced, or oncologic resection inducedcraniofacial defects, and also is useful in cosmetic plastic surgery.

[0077] The polypeptide of this invention may also be used in thetreatment of periodontal disease, and in other tooth repair processes.Such agents may provide an environment to attract bone-forming cells,stimulate growth of bone-forming cells or induce differentiation ofprogenitors of bone-forming cells. The polypeptide of the invention mayalso be useful in the treatment of osteoporosis or osteoarthritis, suchas through stimulation of bone and/or cartilage repair or by blockinginflammation or processes of tissue destruction (collagenase activity,osteoclast activity, etc.) mediated by inflammatory processes.

[0078] Another category of tissue regeneration activity that may beattributable to the polypeptide of the invention is tendon/ligamentformation. A polypeptide of the invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing atendon/Ligament-like tissue inducing polypeptide may have prophylacticuse in preventing damage to tendon or ligament tissue, as well as use inthe improved fixation of tendon or ligament to bone or other tissues,and in repairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of theinvention contributes to the repair of congenital, trauma induced, orother tendon or ligament defects of other origin, and is also useful incosmetic plastic surgery for attachment or repair of tendons orligaments. The compositions of the invention may provide an environmentto attract tendon- or ligament-forming cells, stimulate growth oftendon- or ligament-forming cells, induce differentiation of progenitorsof tendon- or ligament-forming cells, or induce growth of tendonLigament cells or progenitors ex vivo for return in vivo to effecttissue repair. The compositions of the invention may also be useful inthe treatment of tendinitis, carpal tunnel syndrome and other tendon orligament defects. The compositions may also include an appropriatematrix and/or sequestering agent as a carrier as is well known in theart.

[0079] The polypeptide of the invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue. i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies. as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, the polypeptide of the invention maybe used in the treatment of diseases of the peripheral nervous system,such as peripheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using the polypeptide of the invention.

[0080] It is expected that the polypeptide of the invention may alsoexhibit activity for generation of other tissues, such as organs(including, for example, pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac) and vascular(including vascular endothelium) tissue, or for promoting theproliferation of cells comprising such tissues. Part of the desiredeffects may be by inhibition of fibrotic scarring to allow normal tissueto regenerate.

[0081] A polypeptide of the invention may also be useful for gutprotection or regeneration and treatment of lung or liver fibrosis,reperfusion injury in various tissues, and conditions resulting fromsystemic cytokine damage.

[0082] 5) Activin/Inhibin Activity

[0083] The polypeptide of the invention may also exhibit activin- orinhibin-related activities. Inhibins are characterized by their abilityto inhibit the release of follicle stimulating hormone (FSH), whileactivins and are characterized by their ability to stimulate the releaseof follicle stimulating hormone (FSH). Thus, a polypeptide of theinvention alone or in heterodimers with a member of the inhibin αfamily, may be useful as a contraceptive based on the ability ofinhibins to decrease fertility in female mammals and decreasespermatogenesis in male mammals. Administration of sufficient amounts ofother inhibins can induce infertility in these mammals. Alternatively,the polypeptide of the invention, as a homodimer or as a heterodimerwith other protein subunits of the inhibin-β group, may be useful as afertility inducing therapeutic, based upon the ability of activinmolecules in stimulating FSH release from cells of the anteriorpituitary. See for example, U.S. Pat. No. 4,798,885. The polypeptide ofthe invention may also be useful for advancement of the onset offertility in sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs.

[0084] 6) Chemotactic/Chemokinetic Activity

[0085] A polypeptide of the invention may have chemotactic orchemokinetic activity (e.g., act as a chemokine) for mammalian cells,including. for example. monocytes, neutrophils, T-cells, mast cells,eosinophils and/or endothelial cells. Chemotactic and chemokineticproteins can be used to mobilized or attract a desired cell populationto a desired site of action. Chemotactic or chemokinetic proteinsprovide particular advantages in treatment of wounds and other trauma totissues, as well as in treatment of localized infections. For example,attraction of lymphocytes, monocytes or neutrophils to tumors or sitesof infection may result in improved immune responses against the tumoror infecting agent.

[0086] A protein or peptide has chemotactic activity for a particularcell population if it can stimulate, directly or indirectly, thedirected orientation or movement of such cell population. Preferably,the protein or peptide has the ability to directly stimulate directedmovement of cells. Whether a particular protein has chemotactic activityfor a population of cells can be readily determined by employing suchprotein or peptide in any known assay for cell chemotaxis.

[0087] 7) Hemostatic and Thrombolytic Activity

[0088] The polypeptide of the invention may also exhibit hemostatic orthrombolyic activity. As a result, such a protein is expected to beuseful in treatment of various coagulation disorders (includinghereditary disorders, such as hemophilias) or to enhance coagulation andother hemostatic events in treating wounds resulting from trauma,surgery or other causes. A protein of the invention may also be usefulfor dissolving or inhibiting formation of thromboses and for treatmentand prevention of conditions resulting therefrom (such as. for example,infarction or stroke).

[0089] 8) Receptor/Ligand Activity

[0090] The polypeptide of the invention may also demonstrate activity asreceptors, receptor ligands or inhibitors or agonists of receptor/ligandinteractions. Examples of such receptors and ligands include, withoutlimitation, cytokine receptors and their ligands, receptor kinases andtheir ligands, receptor phosphatases and their ligands, receptorsinvolved in cell-cell interactions and their ligands (including cellularadhesion molecules (such as selectins, integrins and their ligands) andreceptor/ligand pairs involved in antigen presentation, antigenrecognition and development of cellular and humoral immune responses).Receptors and ligands are also useful for screening of potential peptideor small molecule inhibitors of the relevant receptor/ligandinteraction. A polypeptide of the invention (including, withoutlimitation, fragments of receptors and ligands) may themselves be usefulas inhibitors of receptor/ligand interactions.

[0091] 9) Other Activity

[0092] The polypeptide of the invention may also exhibit one or more ofthe following additional activities or effects: inhibiting the growth,infection or function of, or killing, infectious agents, including,bacteria, viruses, fungi and other parasites;

[0093] effecting (suppressing or enhancing) bodily characteristics,including, height, weight, hair color, eye color, skin, fat to leanratio or other tissue pigmentation, or organ or body part size or shape(such as, for example, breast augmentation or diminution);

[0094] effecting elimination of dietary fat, protein, carbohydrate;

[0095] effecting behavioral characteristics, including appetite, libido,stress, cognition (including cognitive disorders), depression andviolent behaviors;

[0096] providing analgesic effects or other pain reducing effects;

[0097] promoting differentiation and growth of embryonic stem cells inlineages other than hematopoietic lineages;

[0098] in the case of enzymes, correcting deficiencies of the enzyme andtreating deficiency-related diseases.

[0099] The polypeptide with above activities, is suspected to havefollowing functions by itself or interaction with its ligands orreceptors or association with other molecules. For example,

[0100] proliferation or cell death of B cells, T cells and/or mast cellsor class specific induction of B cells by promotion of class switch ofimmunoglobulin genes; differentiation of B cells to antibody-formingcells; proliferation, differentiation, or cell death of precursors ofgranulocytes; proliferation, differentiation, or cell death ofprecursors of monocytes-macrophages;

[0101] proliferation, of up regulation or cell death of neutrophils,monocytes-macrophages, eosinophils and/or basophils;

[0102] proliferation, or cell death of precursors of megakaryocytes;

[0103] proliferation, differentiation, or cell death of precursors ofneutrophils; proliferation, differentiation, or cell death of precursorsof T cells and B cells; promotion of production of erythrocytes;sustainment of proliferation of erythrocytes, neutrophils, eosinophils,basophils, monocytes-macrophages, mast cells, precursors ofmegakaryocyte; promotion of migration of neutrophils,monocytes-macrophages, B cells and/or T cells;

[0104] proliferation or cell death of thymocytes; suppression ofdifferentiation of adipocytes; proliferation or cell death of naturalkiller cells;

[0105] proliferation or cell death of hematopoietic stem cells;suppression of proliferation of stem cells and each hematopoieticprecursorcells; promotion of differentiation from mesenchymal stem cellsto osteoblasts or chondrocytes, proliferation or cell death ofmesenchymal stem cells, osteoblasts or chondrocytes and promotion ofbone absorption by activation of osteoclasts and promotion ofdifferentiation from monocytes to osteoclasts.

[0106] This peptide is also suspected to function to nervous system, soexpected to have functions below; differentiation to kinds ofneurotransmitter-responsive neurons, survival or cell death of thesecells; promotion of proliferation or cell death of glial cells; spreadof neural dendrites; survival or cell death of gangriocytes;proliferation, promotion of differentiation, or cell death ofastrocytes; proliferation or survival of peripheral neurons;proliferation or cell death of Schwann cells; proliferation, survival orcell death of motoneurons.

[0107] Furthermore, in the process of development of early embryonic,this polypeptide is expected to promote or inhibit the organogenesis ofepidermis, brain, backbone, and nervous system by induction of ectoderm,that of notochord connective tissues(bone, muscle, tendon), hemocytes,heart, kidney, and genital organs by induction of mesoderm, and that ofdigestive apparatus (stomach, intestine, liver, pancreas), respiratoryapparatus (lung, trachea) by induction of endoderm. In adult, also, thispolypeptide is thought to proliferate or inhibit the above organs.

[0108] Therefore, this polypeptide itself is expected to be used as anagent for the prevention or treatment of disease of progression orsuppression of immune, nervous, or bone metabolic function, hypoplasiaor overgrowth of hematopoietic cells: inflammatory disease (rheumatism,ulcerative colitis, etc.), decrease of hematopoietic stem cells afterbone marrow transplantation, decrease of leukocytes, platelets, B-cells,or T-cells after radiation exposure or chemotherapeutic dosage againstcancer or leukemia, anemia, infectious disease, cancer, leukemia, AIDS,bone metabolic disease (osteoporosis etc.), various degenerative disease(Alzheimer's disease, multiple sclerosis, etc.), or nervous lesion.

[0109] In addition, since this polypeptide is thought to induce thedifferentiation or growth of organs derived from ectoderm, mesoderm, andendoderm, this polypeptide is expected to be an agent for tissue repair(epidermis, bone, muscle, tendon, heart, kidney, stomach, intestine,liver, pancreas, lung, and trachea, etc.).

[0110] Quantitation of the polypeptide of the invention in the body canbe performed using polyclonal or monoclonal antibodies against thepolypeptide of the invention. It can be used the study of relationshipbetween this polypeptide and disease or diagnosis of disease, and so on.Polyclonal and monoclonal antibodies can be prepared using thispolypeptide or its fragment as an antigen by conventional methods.

[0111] Identification, purification or molecular cloning of known orunknown proteins which bind the polypeptide of the invention (preferablypolypeptide of extracellular domain) can be performed using thepolypeptide of the invention by, for example, preparation of theaffinity-column.

[0112] Identification of the downstream signal transmission moleculeswhich interact with the polypeptide of the invention in cytoplasma andmolecular cloning of the gene can be performed:

[0113] by west-western method using the polypeptide of the invention(preferably polypeptide of transmembrane region or intracellular domain)or

[0114] by yeast two-hybrid system using the cDNA (preferably cDNAencoding transmembrane region or cytoplasmic domain of the polypeptide).

[0115] Agonists/antagonists of this receptor polypeptide and inhibitorsbetween receptor and signal transduction molecules can be screened usingthe polypeptide of the invention.

[0116] cDNAs of the invention are useful not only the important andessential template for the production of the polypeptide of theinvention which is expected to be largely useful, but also be useful fordiagnosis or therapy (for example, treatment of gene lacking, treatmentto stop the expression of the polypeptide by antisense DNA (RNA)).Genomic DNA may be isolated with the cDNA of the invention, as a probe.As the same manner, a human gene encoding which can be highly homologousto the cDNA of the invention, that is, which encodes a polypeptidehighly homologous to the polypeptide of the invention and a gene ofanimals excluding mouse which can be highly homologous to the cDNA ofthe invention, also may be isolated.

[0117] [Application to Medicaments]

[0118] The polypeptide of the invention or the antibody specific for thepolypeptide of the invention is administered systemically or topicallyand in general orally or parenterally for preventing or treatingdiseases related to incomplete growth or abnormal growth ofhematopoietic system cells, acceleration or reduction of nerve systemfunctions or acceleration or reduction of immune system functions, suchas inflammatory diseases (e.g., rheumatoid, ulcerative colitis, etc.),cytopenia of hematopoietic stem cells after bone marrow transplantation,cytopenia of leukocytes, platelets, B cells or T cells after radiationtreatment or after administration of a chemotherapeutic agent, anemia,infectious diseases, cancer, leukemia, AIDS, and various degenerativediseases (e.g., Alzheimer's disease, multiple sclerosis, etc.), or nervedamage, for preventing or treating metabolic disorder of bones (e.g.,osteoporosis, etc.), or for repairing tissues. Oral administration,intravenous injection and intraventricular administration are preferred.

[0119] The doses to be administered depend upon age, body weight,symptom, desired therapeutic effect, route of administration, andduration of the treatment etc. In human adults, one dose per person isgenerally between 100 μg and 100 mg, by oral administration, up toseveral times per day, and between 10 μg and 100 mg, by parenteraladministration up to several times per day.

[0120] As mentioned above, the doses to be used depend upon variousconditions. Therefore, there are cases in which doses lower than orgreater than the ranges specified above may be used.

[0121] The compounds of the invention, may be administered as solidcompositions, liquid compositions or other compositions for oraladministration, as injections, liniments or suppositories etc. forparenteral administration.

[0122] Solid compositions for oral administration include compressedtablets, pills, capsules, dispersible powders, granules. Capsulesinclude soft or hard capsules.

[0123] In such compositions, one or more of the active compound(s) is orare admixed with at least one inert diluent (such as lactose, mannitol,glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch,polyvinylpyrrolidone, magnesium metasilicate aluminate, etc.). Thecompositions may also comprise, as is normal practice, additionalsubstances other than inert diluents: e.g. lubricating agents (such asmagnesium stearate etc.), disintegrating agents (such as cellulosecalcium glycolate, etc.), stabilizing agents (such as human serumalbumin, lactose etc.), and assisting agents for dissolving (such asarginine, asparaginic acid etc.).

[0124] The tablets or pills may, if desired, be coated with a film ofgastric or enteric materials (such as sugar, gelatin, hydroxypropylcellulose or hydroxypropylmethyl cellulose phthalate, etc.), or becoated with more than two films. And then, coating may includecontainment within capsules of absorbable materials such as gelatin.

[0125] Liquid compositions for oral administration includepharmaceutically-acceptable emulsions, solutions, syrups and elixirs. Insuch compositions, one or more of the active compound(s) is or arecontained in inert diluent(s) commonly used (purified water, ethanoletc.) Besides inert diluents, such compositions may also compriseadjuvants (such as wetting agents, suspending agents, etc.), sweeteningagents, flavoring agents, perfuming agents, and preserving agents.

[0126] Other compositions for oral administration include spraycompositions which may be prepared by known methods and which compriseone or more of the active compound(s). Spray compositions may compriseadditional substances other than inert diluents: e.g. stabilizing agents(sodium sulfite etc.), isotonic buffer (sodium chloride, sodium citrate,citric acid, etc.). For preparation of such spray compositions, forexample, the method described in the U.S. Pat. No. 2,868,691 or3,095,355 (herein incorporated in their entireties by reference) may beused.

[0127] Injections for parenteral administration include sterile aqueousor non-aqueous solutions, suspensions and emulsions. In suchcompositions, one or more active compound(s) is or are admixed with atleast one inert aqueous diluent(s) (distilled water for injection,physiological salt solution, etc.) or inert non-aqueous diluents(s)(propylene glycol, polyethylene glycol, olive oil, ethanol, POLYSOLBATE80 TM , etc.).

[0128] Injections may comprise additional compound other than inertdiluents: e.g. preserving agents, wetting agents, emulsifying agents,dispersing agents, stabilizing agent (such as human serum albumin,lactose, etc.), and assisting agents such as assisting agents fordissolving (arginine, asparaginic acid, etc.).

BEST MODE CARRING OUT THE INVENTION

[0129] The invention are illustrated by the following examples, but notlimit the invention.

EXAMPLE

[0130] Total RNA was prepared from human bone marrow stromal cell lineHAS303 (provided from Professor Keisuke Sotoyama, Dr. Makoto Aizawa,first medicine, Tokyo Medical College; See J. Cell. Physiol.,148:245-251 (1991) and Experimental Hematol., 22:482-487(1994)) byTRIzol reagent (Trade Mark, GIBCOBRL). Poly(A)RNA was purified from thetotal RNA by mRNA purification kit (commercial name, Pharmacia).

[0131] Double strand CDNA was synthesized by Superscript Plasmid Systemfor CDNA Synthesis and Plasmid Cloning (brand name, GIBCOBRL) with abovepoly(A)RNA as template and random 9mer as primer which was containingXhoI site:

[0132] SEQ ID NO. 9

[0133] 5′-CGA TTG AAT TCT AGA CCT GCC TCG AGN NNN NNN NN-3′

[0134] cDNA was ligated EcoRI adapter by DNA ligation kit ver.2 (tradename, Takara Shuzo; this kit was used in all ligating steps hereafter.)and digested by XhoI. cDNAs were separated by agarose-gelelectrophoresis. 300-800 bp cDNAs were isolated and were ligated toEcoRI/NotI site of pSUC2 (see U.S. Pat. No. 5,536,637). E. Coli DH10Bstrain were transformed by pSUC2 with electropolation to obtain yeastSST CDNA library.

[0135] Plasmids of the cDNA library were prepared. Yeast YTK12 strainwere transformed by the plasmids with lithium acetate method (CurrentProtocols In Molecular Biology 13.7.1). The transformed yeast wereplated on triptphan-free medium (CMD-Try medium) for selection. Theplate was incubated for 48 hour at 30° C. Replica of the colony which isobtained by Accutran Replica Plater (trade name, Schleicher & Schuell)were place YPR plate containing raffinose for carbon source, and theplate was incubated for 14 days at 30° C. After 3 days, each colonyappeared was streaked on YPR plate again. The plates were incubated for48 hours at 30° C. Single colony was inoculated to YPR medium and wasincubated for 48 hours at 30° C. Then plasmids were prepared. InsertcDNA was amplified by PCR with two kind primers which exist end side ofcloning site on pSUC2 (sense strand primers were biotinylated).Biotinylated single strand of cDNAs were purified with Dynabeads (tradename, DYNAL) and determined the nucleotide sequences. Sequencing wasperformed by Dye Terminator Cycle Sequencing Ready Reaction with DNASequencing kit (trade name, Applied Biosystems Inc.) and sequence wasdetermined by DNA sequencer 373 (Applied Biosystems Inc.). Allsequencing hereafter was carried with this method.

[0136] The clone named OAF065 is not registered on databases by homologysearch of nucleotide sequence and deduced amino acid sequence and so itis cleared that the sequence is novel one. We confirmed that OAF065contains signal peptide in view of function and structure, by comparisonwith known peptide which has signal peptide and deduced amino acidsequence. Full length cDNA of OAF065 was isolated by 3′-RACE(RapidAmplification of cDNA End). Marathon cDNA Amplification Kit(trade name,Clontech) was used in 3′-RACE. Adaptor-ligated double stranded cDNA wasprepared from poly(A)RNA of HAS303 in line with the method of the kit.OAF065 specific primer F3 (28mer):

[0137] SEQ ID NO. 10

[0138] 5′-AGA AAG ATG GCT TTA AAA GTG CTA CTA G-3′

[0139] which included a deduced initiation ATG coden region based on theinformation of nucleotide sequence by SST was prepared. PCR wasperformed with the said primer and adapter primer attached in the kit.Two kinds of cDNAs (4.0 kb and 1.5 kb) were amlified and 4.0 kb-cDNA wasnamed OAF065α and 1.5 kb-cDNA was named OAF065β.

[0140] Two kinds cDNAs were separated with agarose-gel electrophoresis,and to pT7 Blue-2 T-Vector (trade name, Novagen), ligated in andtransformed to E. Coli DH5α and then plasmid was prepared. Nucleotidesequences of 5′-end were determined, and the existance of nucleotidesequence OAF065 specific primer F3 were confirmed in both nucleotidesequences. 5′-End nucleotide sequence (ca 1.7 kb) of OAF065α and fulllength nucleotide sequence of OAF065β were determined and then obtainedsequences shown in SEQ ID NOs 3 and 7. Open reading frame was searchedand deduced amino acid sequences shown in SEQ ID NO. 1 and 5 wereobtained.

[0141] Compared with the nucleotide sequences of OAF065α and OAF065β,nucletide sequences from 1 to 1290 base were completely same, butsequences downstream from 1291 base had no homology each other. Comparedwith amino acid sequences of OAF065α and OAF065β, amino acids from 1 to415 in N-termini were completely same, only two amino acids in C-terminiof OAF065α were replaced to 8 amino acids (Val Arg Gln Arg Leu Gly SerLeu) in the sequence of OAF065β. It was revealed that OAF065α andOAF065β were novel type-I membrane proteins by hydrophobisity analysisand that the extracellular region and the transmembrane region of bothsequences were consistant.

[0142] The polypeptide OAF065α and OAF065β of the invention are notknown one, when amino acid sequences of the polypeptide was compared bya computer to all known sequences in data base of Swiss Prot Release 33.Extracellular Cys rich region which commonly exists in the TNF receptorfamily was identfied in the polypeptide of the invention.

[0143] That is, compared with amino acid sequences of the polypeptide ofthe invention (OAF065s) and other members of TNF receptor family i.e.human necrosis factor receptor 1 (hTNFR1), human necrosis factorreceptor 2 (hTNFR2), human nerve growth factor receptor (hNGFR), andhuman Fas (hFas), it was revealed that the polypeptides (OAF065s) of theinvention are type-I membrane protein and they have extracellular Cysrich region which commonly exists in the TNF (Tumor necrosis factor)receptor family in FIG. 1.

[0144] Therefore, it was confirmed that the polypeptides OAF065α andOAF065β of the invention are novel membrane proteins which belong to theTNF receptor family.

1 10 1 1269 DNA Homo sapiens 1 atggctttaa aagtgctact agaacaagagaaaacgtttt tcactctttt agtattacta 60 ggctatttgt catgtaaagt gacttgtgaaacaggagact gtagacagca agaattcagg 120 gatcggtctg gaaactgtgt tccctgcaaccagtgtgggc caggcatgga gttgtctaag 180 gaatgtggct tcggctatgg ggaggatgcacagtgtgtga cgtgccggct gcacaggttc 240 aaggaggact ggggcttcca gaaatgcaagccctgtctgg actgcgcagt ggtgaaccgc 300 tttcagaagg caaattgttc agccaccagtgatgccatct gcggggactg cttgccagga 360 ttttatagga agacgaaact tgtcggctttcaagacatgg agtgtgtgcc ttgtggagac 420 cctcctcctc cttacgaacc gcactgtgccagcaaggtca acctcgtgaa gatcgcgtcc 480 acggcctcca gcccacggga cacggcgctggctgccgtta tctgcagcgc tctggccacc 540 gtcctgctgg ccctgctcat cctctgtgtcatctattgta agagacagtt tatggagaag 600 aaacccagct ggtctctgcg gtcacaggacattcagtaca acggctctga gctgtcgtgt 660 cttgacagac ctcagctcca cgaatatgcccacagagcct gctgccagtg ccgccgtgac 720 tcagtgcaga cctgcgggcc ggtgcgcttgctcccatcca tgtgctgtga ggaggcctgc 780 agccccaacc cggcgactct tggttgtggggtgcattctg cagccagtct tcaggcaaga 840 aacgcaggcc cagccgggga gatggtgccgactttcttcg gatccctcac gcagtccatc 900 tgtggcgagt tttcagatgc ctggcctctgatgcagaatc ccatgggtgg tgacaacatc 960 tctttttgtg actcttatcc tgaactcactggagaagaca ttcattctct caatccagaa 1020 cttgaaagct caacgtcttt ggattcaaatagcagtcaag atttggttgg tggggctgtt 1080 ccagtccagt ctcattctga aaactttacagcagctactg atttatctag atataacaac 1140 acactggtag aatcagcatc aactcaggatgcactaacta tgagaagcca gctagatcag 1200 gagagtggcg ctatcatcca cccagccactcagacgtccc tccaggtaag gcagcgactg 1260 ggttccctg 1269 2 1704 DNA Homosapiens 2 gggaacgtag aactctccaa caataaatac atttgataag aaagatggctttaaaagtgc 60 tactagaaca agagaaaacg tttttcactc ttttagtatt actaggctatttgtcatgta 120 aagtgacttg tgaaacagga gactgtagac agcaagaatt cagggatcggtctggaaact 180 gtgttccctg caaccagtgt gggccaggca tggagttgtc taaggaatgtggcttcggct 240 atggggagga tgcacagtgt gtgacgtgcc ggctgcacag gttcaaggaggactggggct 300 tccagaaatg caagccctgt ctggactgcg cagtggtgaa ccgctttcagaaggcaaatt 360 gttcagccac cagtgatgcc atctgcgggg actgcttgcc aggattttataggaagacga 420 aacttgtcgg ctttcaagac atggagtgtg tgccttgtgg agaccctcctcctccttacg 480 aaccgcactg tgccagcaag gtcaacctcg tgaagatcgc gtccacggcctccagcccac 540 gggacacggc gctggctgcc gttatctgca gcgctctggc caccgtcctgctggccctgc 600 tcatcctctg tgtcatctat tgtaagagac agtttatgga gaagaaacccagctggtctc 660 tgcggtcaca ggacattcag tacaacggct ctgagctgtc gtgtcttgacagacctcagc 720 tccacgaata tgcccacaga gcctgctgcc agtgccgccg tgactcagtgcagacctgcg 780 ggccggtgcg cttgctccca tccatgtgct gtgaggaggc ctgcagccccaacccggcga 840 ctcttggttg tggggtgcat tctgcagcca gtcttcaggc aagaaacgcaggcccagccg 900 gggagatggt gccgactttc ttcggatccc tcacgcagtc catctgtggcgagttttcag 960 atgcctggcc tctgatgcag aatcccatgg gtggtgacaa catctctttttgtgactctt 1020 atcctgaact cactggagaa gacattcatt ctctcaatcc agaacttgaaagctcaacgt 1080 ctttggattc aaatagcagt caagatttgg ttggtggggc tgttccagtccagtctcatt 1140 ctgaaaactt tacagcagct actgatttat ctagatataa caacacactggtagaatcag 1200 catcaactca ggatgcacta actatgagaa gccagctaga tcaggagagtggcgctatca 1260 tccacccagc cactcagacg tccctccagg aagcttaaag aacctgcttctttctgcagt 1320 agaagcgtgt gctggaaccc aaagagtact cctttgttag gcttatggactgagcagtct 1380 ggaccttgca tggcttctgg ggcaaaaata aatctgaacc aaactgacggcatttgaagc 1440 ctttcagcca gttgcttctg agccagacca gctgtaagct gaaacctcaatgaataacaa 1500 gaaaagactc caggccgact catgatactc tgcatctttc ctacatgagaagcttctctg 1560 ccacaaaagt gacttcaaag acggatgggt tgagctggca gcctatgagattgtggacat 1620 ataacaagaa acagaaatgc cctcatgctt attttcatgg tgattgtggttttacaagac 1680 tgaagaccca gagtatactt tttc 1704 3 1704 DNA Homo sapiensmisc_feature Origin human bone marrow stromal cell line HAS303 3gggaacgtag aactctccaa caataaatac atttgataag aaag atg gct tta aaa 56 MetAla Leu Lys -25 gtg cta cta gaa caa gag aaa acg ttt ttc act ctt tta gtatta cta 104 Val Leu Leu Glu Gln Glu Lys Thr Phe Phe Thr Leu Leu Val LeuLeu -20 -15 -10 ggc tat ttg tca tgt aaa gtg act tgt gaa aca gga gac tgtaga cag 152 Gly Tyr Leu Ser Cys Lys Val Thr Cys Glu Thr Gly Asp Cys ArgGln -5 -1 1 5 10 caa gaa ttc agg gat cgg tct gga aac tgt gtt ccc tgc aaccag tgt 200 Gln Glu Phe Arg Asp Arg Ser Gly Asn Cys Val Pro Cys Asn GlnCys 15 20 25 ggg cca ggc atg gag ttg tct aag gaa tgt ggc ttc ggc tat ggggag 248 Gly Pro Gly Met Glu Leu Ser Lys Glu Cys Gly Phe Gly Tyr Gly Glu30 35 40 gat gca cag tgt gtg acg tgc cgg ctg cac agg ttc aag gag gac tgg296 Asp Ala Gln Cys Val Thr Cys Arg Leu His Arg Phe Lys Glu Asp Trp 4550 55 ggc ttc cag aaa tgc aag ccc tgt ctg gac tgc gca gtg gtg aac cgc344 Gly Phe Gln Lys Cys Lys Pro Cys Leu Asp Cys Ala Val Val Asn Arg 6065 70 75 ttt cag aag gca aat tgt tca gcc acc agt gat gcc atc tgc ggg gac392 Phe Gln Lys Ala Asn Cys Ser Ala Thr Ser Asp Ala Ile Cys Gly Asp 8085 90 tgc ttg cca gga ttt tat agg aag acg aaa ctt gtc ggc ttt caa gac440 Cys Leu Pro Gly Phe Tyr Arg Lys Thr Lys Leu Val Gly Phe Gln Asp 95100 105 atg gag tgt gtg cct tgt gga gac cct cct cct cct tac gaa ccg cac488 Met Glu Cys Val Pro Cys Gly Asp Pro Pro Pro Pro Tyr Glu Pro His 110115 120 tgt gcc agc aag gtc aac ctc gtg aag atc gcg tcc acg gcc tcc agc536 Cys Ala Ser Lys Val Asn Leu Val Lys Ile Ala Ser Thr Ala Ser Ser 125130 135 cca cgg gac acg gcg ctg gct gcc gtt atc tgc agc gct ctg gcc acc584 Pro Arg Asp Thr Ala Leu Ala Ala Val Ile Cys Ser Ala Leu Ala Thr 140145 150 155 gtc ctg ctg gcc ctg ctc atc ctc tgt gtc atc tat tgt aag agacag 632 Val Leu Leu Ala Leu Leu Ile Leu Cys Val Ile Tyr Cys Lys Arg Gln160 165 170 ttt atg gag aag aaa ccc agc tgg tct ctg cgg tca cag gac attcag 680 Phe Met Glu Lys Lys Pro Ser Trp Ser Leu Arg Ser Gln Asp Ile Gln175 180 185 tac aac ggc tct gag ctg tcg tgt ctt gac aga cct cag ctc cacgaa 728 Tyr Asn Gly Ser Glu Leu Ser Cys Leu Asp Arg Pro Gln Leu His Glu190 195 200 tat gcc cac aga gcc tgc tgc cag tgc cgc cgt gac tca gtg cagacc 776 Tyr Ala His Arg Ala Cys Cys Gln Cys Arg Arg Asp Ser Val Gln Thr205 210 215 tgc ggg ccg gtg cgc ttg ctc cca tcc atg tgc tgt gag gag gcctgc 824 Cys Gly Pro Val Arg Leu Leu Pro Ser Met Cys Cys Glu Glu Ala Cys220 225 230 235 agc ccc aac ccg gcg act ctt ggt tgt ggg gtg cat tct gcagcc agt 872 Ser Pro Asn Pro Ala Thr Leu Gly Cys Gly Val His Ser Ala AlaSer 240 245 250 ctt cag gca aga aac gca ggc cca gcc ggg gag atg gtg ccgact ttc 920 Leu Gln Ala Arg Asn Ala Gly Pro Ala Gly Glu Met Val Pro ThrPhe 255 260 265 ttc gga tcc ctc acg cag tcc atc tgt ggc gag ttt tca gatgcc tgg 968 Phe Gly Ser Leu Thr Gln Ser Ile Cys Gly Glu Phe Ser Asp AlaTrp 270 275 280 cct ctg atg cag aat ccc atg ggt ggt gac aac atc tct ttttgt gac 1016 Pro Leu Met Gln Asn Pro Met Gly Gly Asp Asn Ile Ser Phe CysAsp 285 290 295 tct tat cct gaa ctc act gga gaa gac att cat tct ctc aatcca gaa 1064 Ser Tyr Pro Glu Leu Thr Gly Glu Asp Ile His Ser Leu Asn ProGlu 300 305 310 315 ctt gaa agc tca acg tct ttg gat tca aat agc agt caagat ttg gtt 1112 Leu Glu Ser Ser Thr Ser Leu Asp Ser Asn Ser Ser Gln AspLeu Val 320 325 330 ggt ggg gct gtt cca gtc cag tct cat tct gaa aac tttaca gca gct 1160 Gly Gly Ala Val Pro Val Gln Ser His Ser Glu Asn Phe ThrAla Ala 335 340 345 act gat tta tct aga tat aac aac aca ctg gta gaa tcagca tca act 1208 Thr Asp Leu Ser Arg Tyr Asn Asn Thr Leu Val Glu Ser AlaSer Thr 350 355 360 cag gat gca cta act atg aga agc cag cta gat cag gagagt ggc gct 1256 Gln Asp Ala Leu Thr Met Arg Ser Gln Leu Asp Gln Glu SerGly Ala 365 370 375 atc atc cac cca gcc act cag acg tcc ctc cag gaa gcttaaagaacct 1305 Ile Ile His Pro Ala Thr Gln Thr Ser Leu Gln Glu Ala 380385 390 gcttctttct gcagtagaag cgtgtgctgg aacccaaaga gtactcctttgttaggctta 1365 tggactgagc agtctggacc ttgcatggct tctggggcaa aaataaatctgaaccaaact 1425 gacggcattt gaagcctttc agccagttgc ttctgagcca gaccagctgtaagctgaaac 1485 ctcaatgaat aacaagaaaa gactccaggc cgactcatga tactctgcatctttcctaca 1545 tgagaagctt ctctgccaca aaagtgactt caaagacgga tgggttgagctggcagccta 1605 tgagattgtg gacatataac aagaaacaga aatgccctca tgcttattttcatggtgatt 1665 gtggttttac aagactgaag acccagagta tactttttc 1704 4 417PRT Homo sapiens misc_feature Origin human bone marrow stromal cell lineHAS303 4 Met Ala Leu Lys Val Leu Leu Glu Gln Glu Lys Thr Phe Phe Thr Leu-25 -20 -15 -10 Leu Val Leu Leu Gly Tyr Leu Ser Cys Lys Val Thr Cys GluThr Gly -5 -1 1 5 Asp Cys Arg Gln Gln Glu Phe Arg Asp Arg Ser Gly AsnCys Val Pro 10 15 20 Cys Asn Gln Cys Gly Pro Gly Met Glu Leu Ser Lys GluCys Gly Phe 25 30 35 Gly Tyr Gly Glu Asp Ala Gln Cys Val Thr Cys Arg LeuHis Arg Phe 40 45 50 55 Lys Glu Asp Trp Gly Phe Gln Lys Cys Lys Pro CysLeu Asp Cys Ala 60 65 70 Val Val Asn Arg Phe Gln Lys Ala Asn Cys Ser AlaThr Ser Asp Ala 75 80 85 Ile Cys Gly Asp Cys Leu Pro Gly Phe Tyr Arg LysThr Lys Leu Val 90 95 100 Gly Phe Gln Asp Met Glu Cys Val Pro Cys GlyAsp Pro Pro Pro Pro 105 110 115 Tyr Glu Pro His Cys Ala Ser Lys Val AsnLeu Val Lys Ile Ala Ser 120 125 130 135 Thr Ala Ser Ser Pro Arg Asp ThrAla Leu Ala Ala Val Ile Cys Ser 140 145 150 Ala Leu Ala Thr Val Leu LeuAla Leu Leu Ile Leu Cys Val Ile Tyr 155 160 165 Cys Lys Arg Gln Phe MetGlu Lys Lys Pro Ser Trp Ser Leu Arg Ser 170 175 180 Gln Asp Ile Gln TyrAsn Gly Ser Glu Leu Ser Cys Leu Asp Arg Pro 185 190 195 Gln Leu His GluTyr Ala His Arg Ala Cys Cys Gln Cys Arg Arg Asp 200 205 210 215 Ser ValGln Thr Cys Gly Pro Val Arg Leu Leu Pro Ser Met Cys Cys 220 225 230 GluGlu Ala Cys Ser Pro Asn Pro Ala Thr Leu Gly Cys Gly Val His 235 240 245Ser Ala Ala Ser Leu Gln Ala Arg Asn Ala Gly Pro Ala Gly Glu Met 250 255260 Val Pro Thr Phe Phe Gly Ser Leu Thr Gln Ser Ile Cys Gly Glu Phe 265270 275 Ser Asp Ala Trp Pro Leu Met Gln Asn Pro Met Gly Gly Asp Asn Ile280 285 290 295 Ser Phe Cys Asp Ser Tyr Pro Glu Leu Thr Gly Glu Asp IleHis Ser 300 305 310 Leu Asn Pro Glu Leu Glu Ser Ser Thr Ser Leu Asp SerAsn Ser Ser 315 320 325 Gln Asp Leu Val Gly Gly Ala Val Pro Val Gln SerHis Ser Glu Asn 330 335 340 Phe Thr Ala Ala Thr Asp Leu Ser Arg Tyr AsnAsn Thr Leu Val Glu 345 350 355 Ser Ala Ser Thr Gln Asp Ala Leu Thr MetArg Ser Gln Leu Asp Gln 360 365 370 375 Glu Ser Gly Ala Ile Ile His ProAla Thr Gln Thr Ser Leu Gln Glu 380 385 390 Ala 5 1269 DNA Homo sapiens5 atggctttaa aagtgctact agaacaagag aaaacgtttt tcactctttt agtattacta 60ggctatttgt catgtaaagt gacttgtgaa acaggagact gtagacagca agaattcagg 120gatcggtctg gaaactgtgt tccctgcaac cagtgtgggc caggcatgga gttgtctaag 180gaatgtggct tcggctatgg ggaggatgca cagtgtgtga cgtgccggct gcacaggttc 240aaggaggact ggggcttcca gaaatgcaag ccctgtctgg actgcgcagt ggtgaaccgc 300tttcagaagg caaattgttc agccaccagt gatgccatct gcggggactg cttgccagga 360ttttatagga agacgaaact tgtcggcttt caagacatgg agtgtgtgcc ttgtggagac 420cctcctcctc cttacgaacc gcactgtgcc agcaaggtca acctcgtgaa gatcgcgtcc 480acggcctcca gcccacggga cacggcgctg gctgccgtta tctgcagcgc tctggccacc 540gtcctgctgg ccctgctcat cctctgtgtc atctattgta agagacagtt tatggagaag 600aaacccagct ggtctctgcg gtcacaggac attcagtaca acggctctga gctgtcgtgt 660cttgacagac ctcagctcca cgaatatgcc cacagagcct gctgccagtg ccgccgtgac 720tcagtgcaga cctgcgggcc ggtgcgcttg ctcccatcca tgtgctgtga ggaggcctgc 780agccccaacc cggcgactct tggttgtggg gtgcattctg cagccagtct tcaggcaaga 840aacgcaggcc cagccgggga gatggtgccg actttcttcg gatccctcac gcagtccatc 900tgtggcgagt tttcagatgc ctggcctctg atgcagaatc ccatgggtgg tgacaacatc 960tctttttgtg actcttatcc tgaactcact ggagaagaca ttcattctct caatccagaa 1020cttgaaagct caacgtcttt ggattcaaat agcagtcaag atttggttgg tggggctgtt 1080ccagtccagt ctcattctga aaactttaca gcagctactg atttatctag atataacaac 1140acactggtag aatcagcatc aactcaggat gcactaacta tgagaagcca gctagatcag 1200gagagtggcg ctatcatcca cccagccact cagacgtccc tccaggtaag gcagcgactg 1260ggttccctg 1269 6 1496 DNA Homo sapiens 6 gggaacgtag aactctccaacaataaatac atttgataag aaagatggct ttaaaagtgc 60 tactagaaca agagaaaacgtttttcactc ttttagtatt actaggctat ttgtcatgta 120 aagtgacttg tgaaacaggagactgtagac agcaagaatt cagggatcgg tctggaaact 180 gtgttccctg caaccagtgtgggccaggca tggagttgtc taaggaatgt ggcttcggct 240 atggggagga tgcacagtgtgtgacgtgcc ggctgcacag gttcaaggag gactggggct 300 tccagaaatg caagccctgtctggactgcg cagtggtgaa ccgctttcag aaggcaaatt 360 gttcagccac cagtgatgccatctgcgggg actgcttgcc aggattttat aggaagacga 420 aacttgtcgg ctttcaagacatggagtgtg tgccttgtgg agaccctcct cctccttacg 480 aaccgcactg tgccagcaaggtcaacctcg tgaagatcgc gtccacggcc tccagcccac 540 gggacacggc gctggctgccgttatctgca gcgctctggc caccgtcctg ctggccctgc 600 tcatcctctg tgtcatctattgtaagagac agtttatgga gaagaaaccc agctggtctc 660 tgcggtcaca ggacattcagtacaacggct ctgagctgtc gtgtcttgac agacctcagc 720 tccacgaata tgcccacagagcctgctgcc agtgccgccg tgactcagtg cagacctgcg 780 ggccggtgcg cttgctcccatccatgtgct gtgaggaggc ctgcagcccc aacccggcga 840 ctcttggttg tggggtgcattctgcagcca gtcttcaggc aagaaacgca ggcccagccg 900 gggagatggt gccgactttcttcggatccc tcacgcagtc catctgtggc gagttttcag 960 atgcctggcc tctgatgcagaatcccatgg gtggtgacaa catctctttt tgtgactctt 1020 atcctgaact cactggagaagacattcatt ctctcaatcc agaacttgaa agctcaacgt 1080 ctttggattc aaatagcagtcaagatttgg ttggtggggc tgttccagtc cagtctcatt 1140 ctgaaaactt tacagcagctactgatttat ctagatataa caacacactg gtagaatcag 1200 catcaactca ggatgcactaactatgagaa gccagctaga tcaggagagt ggcgctatca 1260 tccacccagc cactcagacgtccctccagg taaggcagcg actgggttcc ctgtgaacac 1320 agcactgact tacagtagatcagaactctg ttcccagcat aagatttggg ggaacctgat 1380 gagttttttt tttgcatctttaataatttc ttgtatgttg tagagtatgt tttaaaataa 1440 atttcaagta ttttttttaaaaactaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1496 7 1496 DNA Homo sapiensmisc_feature Origin human bone marrow stromal cell line HAS303 7gggaacgtag aactctccaa caataaatac atttgataag aaag atg gct tta aaa 56 MetAla Leu Lys -25 gtg cta cta gaa caa gag aaa acg ttt ttc act ctt tta gtatta cta 104 Val Leu Leu Glu Gln Glu Lys Thr Phe Phe Thr Leu Leu Val LeuLeu -20 -15 -10 ggc tat ttg tca tgt aaa gtg act tgt gaa aca gga gac tgtaga cag 152 Gly Tyr Leu Ser Cys Lys Val Thr Cys Glu Thr Gly Asp Cys ArgGln -5 -1 1 5 10 caa gaa ttc agg gat cgg tct gga aac tgt gtt ccc tgc aaccag tgt 200 Gln Glu Phe Arg Asp Arg Ser Gly Asn Cys Val Pro Cys Asn GlnCys 15 20 25 ggg cca ggc atg gag ttg tct aag gaa tgt ggc ttc ggc tat ggggag 248 Gly Pro Gly Met Glu Leu Ser Lys Glu Cys Gly Phe Gly Tyr Gly Glu30 35 40 gat gca cag tgt gtg acg tgc cgg ctg cac agg ttc aag gag gac tgg296 Asp Ala Gln Cys Val Thr Cys Arg Leu His Arg Phe Lys Glu Asp Trp 4550 55 ggc ttc cag aaa tgc aag ccc tgt ctg gac tgc gca gtg gtg aac cgc344 Gly Phe Gln Lys Cys Lys Pro Cys Leu Asp Cys Ala Val Val Asn Arg 6065 70 75 ttt cag aag gca aat tgt tca gcc acc agt gat gcc atc tgc ggg gac392 Phe Gln Lys Ala Asn Cys Ser Ala Thr Ser Asp Ala Ile Cys Gly Asp 8085 90 tgc ttg cca gga ttt tat agg aag acg aaa ctt gtc ggc ttt caa gac440 Cys Leu Pro Gly Phe Tyr Arg Lys Thr Lys Leu Val Gly Phe Gln Asp 95100 105 atg gag tgt gtg cct tgt gga gac cct cct cct cct tac gaa ccg cac488 Met Glu Cys Val Pro Cys Gly Asp Pro Pro Pro Pro Tyr Glu Pro His 110115 120 tgt gcc agc aag gtc aac ctc gtg aag atc gcg tcc acg gcc tcc agc536 Cys Ala Ser Lys Val Asn Leu Val Lys Ile Ala Ser Thr Ala Ser Ser 125130 135 cca cgg gac acg gcg ctg gct gcc gtt atc tgc agc gct ctg gcc acc584 Pro Arg Asp Thr Ala Leu Ala Ala Val Ile Cys Ser Ala Leu Ala Thr 140145 150 155 gtc ctg ctg gcc ctg ctc atc ctc tgt gtc atc tat tgt aag agacag 632 Val Leu Leu Ala Leu Leu Ile Leu Cys Val Ile Tyr Cys Lys Arg Gln160 165 170 ttt atg gag aag aaa ccc agc tgg tct ctg cgg tca cag gac attcag 680 Phe Met Glu Lys Lys Pro Ser Trp Ser Leu Arg Ser Gln Asp Ile Gln175 180 185 tac aac ggc tct gag ctg tcg tgt ctt gac aga cct cag ctc cacgaa 728 Tyr Asn Gly Ser Glu Leu Ser Cys Leu Asp Arg Pro Gln Leu His Glu190 195 200 tat gcc cac aga gcc tgc tgc cag tgc cgc cgt gac tca gtg cagacc 776 Tyr Ala His Arg Ala Cys Cys Gln Cys Arg Arg Asp Ser Val Gln Thr205 210 215 tgc ggg ccg gtg cgc ttg ctc cca tcc atg tgc tgt gag gag gcctgc 824 Cys Gly Pro Val Arg Leu Leu Pro Ser Met Cys Cys Glu Glu Ala Cys220 225 230 235 agc ccc aac ccg gcg act ctt ggt tgt ggg gtg cat tct gcagcc agt 872 Ser Pro Asn Pro Ala Thr Leu Gly Cys Gly Val His Ser Ala AlaSer 240 245 250 ctt cag gca aga aac gca ggc cca gcc ggg gag atg gtg ccgact ttc 920 Leu Gln Ala Arg Asn Ala Gly Pro Ala Gly Glu Met Val Pro ThrPhe 255 260 265 ttc gga tcc ctc acg cag tcc atc tgt ggc gag ttt tca gatgcc tgg 968 Phe Gly Ser Leu Thr Gln Ser Ile Cys Gly Glu Phe Ser Asp AlaTrp 270 275 280 cct ctg atg cag aat ccc atg ggt ggt gac aac atc tct ttttgt gac 1016 Pro Leu Met Gln Asn Pro Met Gly Gly Asp Asn Ile Ser Phe CysAsp 285 290 295 tct tat cct gaa ctc act gga gaa gac att cat tct ctc aatcca gaa 1064 Ser Tyr Pro Glu Leu Thr Gly Glu Asp Ile His Ser Leu Asn ProGlu 300 305 310 315 ctt gaa agc tca acg tct ttg gat tca aat agc agt caagat ttg gtt 1112 Leu Glu Ser Ser Thr Ser Leu Asp Ser Asn Ser Ser Gln AspLeu Val 320 325 330 ggt ggg gct gtt cca gtc cag tct cat tct gaa aac tttaca gca gct 1160 Gly Gly Ala Val Pro Val Gln Ser His Ser Glu Asn Phe ThrAla Ala 335 340 345 act gat tta tct aga tat aac aac aca ctg gta gaa tcagca tca act 1208 Thr Asp Leu Ser Arg Tyr Asn Asn Thr Leu Val Glu Ser AlaSer Thr 350 355 360 cag gat gca cta act atg aga agc cag cta gat cag gagagt ggc gct 1256 Gln Asp Ala Leu Thr Met Arg Ser Gln Leu Asp Gln Glu SerGly Ala 365 370 375 atc atc cac cca gcc act cag acg tcc ctc cag gta aggcag cga ctg 1304 Ile Ile His Pro Ala Thr Gln Thr Ser Leu Gln Val Arg GlnArg Leu 380 385 390 395 ggt tcc ctg tgaacacagc actgacttac agtagatcagaactctgttc 1353 Gly Ser Leu ccagcataag atttggggga acctgatgag ttttttttttgcatctttaa taatttcttg 1413 tatgttgtag agtatgtttt aaaataaatt tcaagtattttttttaaaaa ctaaaaaaaa 1473 aaaaaaaaaa aaaaaaaaaa aaa 1496 8 423 PRT Homosapiens misc_feature Origin human bone marrow stromal cell line HAS303 8Met Ala Leu Lys Val Leu Leu Glu Gln Glu Lys Thr Phe Phe Thr Leu -25 -20-15 -10 Leu Val Leu Leu Gly Tyr Leu Ser Cys Lys Val Thr Cys Glu Thr Gly-5 -1 1 5 Asp Cys Arg Gln Gln Glu Phe Arg Asp Arg Ser Gly Asn Cys ValPro 10 15 20 Cys Asn Gln Cys Gly Pro Gly Met Glu Leu Ser Lys Glu Cys GlyPhe 25 30 35 Gly Tyr Gly Glu Asp Ala Gln Cys Val Thr Cys Arg Leu His ArgPhe 40 45 50 55 Lys Glu Asp Trp Gly Phe Gln Lys Cys Lys Pro Cys Leu AspCys Ala 60 65 70 Val Val Asn Arg Phe Gln Lys Ala Asn Cys Ser Ala Thr SerAsp Ala 75 80 85 Ile Cys Gly Asp Cys Leu Pro Gly Phe Tyr Arg Lys Thr LysLeu Val 90 95 100 Gly Phe Gln Asp Met Glu Cys Val Pro Cys Gly Asp ProPro Pro Pro 105 110 115 Tyr Glu Pro His Cys Ala Ser Lys Val Asn Leu ValLys Ile Ala Ser 120 125 130 135 Thr Ala Ser Ser Pro Arg Asp Thr Ala LeuAla Ala Val Ile Cys Ser 140 145 150 Ala Leu Ala Thr Val Leu Leu Ala LeuLeu Ile Leu Cys Val Ile Tyr 155 160 165 Cys Lys Arg Gln Phe Met Glu LysLys Pro Ser Trp Ser Leu Arg Ser 170 175 180 Gln Asp Ile Gln Tyr Asn GlySer Glu Leu Ser Cys Leu Asp Arg Pro 185 190 195 Gln Leu His Glu Tyr AlaHis Arg Ala Cys Cys Gln Cys Arg Arg Asp 200 205 210 215 Ser Val Gln ThrCys Gly Pro Val Arg Leu Leu Pro Ser Met Cys Cys 220 225 230 Glu Glu AlaCys Ser Pro Asn Pro Ala Thr Leu Gly Cys Gly Val His 235 240 245 Ser AlaAla Ser Leu Gln Ala Arg Asn Ala Gly Pro Ala Gly Glu Met 250 255 260 ValPro Thr Phe Phe Gly Ser Leu Thr Gln Ser Ile Cys Gly Glu Phe 265 270 275Ser Asp Ala Trp Pro Leu Met Gln Asn Pro Met Gly Gly Asp Asn Ile 280 285290 295 Ser Phe Cys Asp Ser Tyr Pro Glu Leu Thr Gly Glu Asp Ile His Ser300 305 310 Leu Asn Pro Glu Leu Glu Ser Ser Thr Ser Leu Asp Ser Asn SerSer 315 320 325 Gln Asp Leu Val Gly Gly Ala Val Pro Val Gln Ser His SerGlu Asn 330 335 340 Phe Thr Ala Ala Thr Asp Leu Ser Arg Tyr Asn Asn ThrLeu Val Glu 345 350 355 Ser Ala Ser Thr Gln Asp Ala Leu Thr Met Arg SerGln Leu Asp Gln 360 365 370 375 Glu Ser Gly Ala Ile Ile His Pro Ala ThrGln Thr Ser Leu Gln Val 380 385 390 Arg Gln Arg Leu Gly Ser Leu 395 9 35DNA Artificial Primer 9 cgattgaatt ctagacctgc ctcgagnnnn nnnnn 35 10 28DNA Artificial Primer 0AF065 10 agaaagatgg ctttaaaagt gctactag 28

1. Substantially purified form of the polypeptide that comprising theamino-acid sequence shown in SEQ ID NO. 1 or 5, homologue thereof,fragment thereof or homologue of the fragment.
 2. A polypeptideaccording to claim 1 that comprising the amino-acid sequence shown inSEQ ID NO. 1 or
 5. 3. A cDNA encoding the polypeptide according toclaim
 1. 4. A cDNA according to claim 3 that comprising the nucleotidesequence shown in SEQ ID NO. 2 or 6 or a fragment cDNA selectivelyhybridized to the cDNA.
 5. A cDNA according to claim 3 that comprisingthe nucleotide sequence shown in SEQ ID NO. 3 or 8 or a fragment cDNAselectively hybridized to the cDNA.
 6. A replication or expressionvector carrying the cDNA according to claim 3 to
 5. 7. A host celltransformed with the replication or expression vector according to claim6.
 8. A method for producing the polypeptide according to claim 1 or 2which comprises culturing a host cell according to claim 7 under acondition effective to express the polypeptide according to claim 1 or2.
 9. A monoclonal or polyclonal antibody against the polypeptideaccording to claim 1 or
 2. 10. A pharmaceutical composition containingthe polypeptide according to claim 1 or 2 or the antibody according toclaim 9, in association with pharmaceutically acceptable diluent and/orcarrier.